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. Author manuscript; available in PMC: 2019 Sep 15.
Published in final edited form as: J Immunol. 2018 Jul 30;201(6):1705–1716. doi: 10.4049/jimmunol.1800202

Figure 3. Cybb controls Caspase1 activation in macrophages and dendritic cells during Mtb infection.

Figure 3.

Bone marrow-derived macrophages (BMDMs) or Bone marrow-derived dendritic cells (BMDCs) from wild type or Cybb−/− mice were left untreated or infected with Mtb for 4 hours then washed with fresh media. 18 hours later supernatants were harvested and the levels of A. and B. IL-1β and C. and D. TNFα were quantified in the supernatants by ELISA. Shown is the mean of 4 biological replicates normalized to a standard curve +/− s.d. *** p<.001 by unpaired two-tailed t-test. E. and F. Viability of remaining cells was determined by quantifying ATP via luminescence and compared to cells at 4 hours post-infection (mean % viability +/− s.d.). Data in A, C and E are representative of five independent experiments with at least 3 biological replicates per experiment. Data in B, D and F are representative of three independent experiments with 4 biological replicates per experiment. G. Relative RNA expression of Il1b (compared to β-Actin) was determined from wild type and Cybb−/− BMDMs left untreated or infected for 24 hours with Mtb (mean +/− s.d.) by qRT-PCR. Data are representative of two independent experiments with 3–4 biological replicates per group. H. Immunoblot analysis was used to assess the activation of Caspase 1 from Wild type and Cybb−/− BMDMs infected for 24 hours with Mtb. Data are representative of 3 independent experiments with at least 3 biological replicates analyzed per experiment. I. Immunoblots were quantified by comparing the intensity of activated p20-Caspase 1 to total pro-Caspase1 bands. Quantification was done on three biological replicates. * p<.05 by unpaired two-tailed t-test. J. Relative RNA expression of Il1b (compared to Actb) was determined from wild type and Cybb−/− BMDMs left untreated or treated with Pam3CSK4 for 24 hours (mean +/− s.d.) by qRT-PCR. Data are representative of two independent experiments with 3–4 biological replicates per group. K. Wild type and Cybb−/− BMDMs were left untreated or treated with PAM3CSK4 for 12 hours, supernatants were harvested and the levels of IL-1β were quantified by ELISA (mean +/− s.d.). ** p<.01 by unpaired two-tailed t-test. Data are representative of three independent experiments with 4 biological replicates per experiment.